Zen 2010 b sp1

HuH-7 cells were cultured in glass-bottomed dishes (Matsunami Glass, Osaka, Japan) in a chamber unit (INUG2-ZIL; Tokai Hit, Hamamatsu, Shizuoka, Japan) equipped to the LSM700 inverted laser-scanning confocal fluorescence microscope. Time-series data of fluorescence and DIC images was obtained for Ca²⁺ and mitochondrial superoxide measurements using a fluorogenic dye, Fluo-4 AM (Dojindo, Kumamoto, Japan), or MitoSOX™ Red (Thermo Fisher Scientific), respectively. Fluo-4 experiments were performed after GGA treatment in serum-free DMEM or serum/Ca²⁺-free DMEM. The mean pixel intensity of Fluo-4 fluorescence for each cell was determined in regions of interest (ROIs) of the time-lapse image series using Zen 2010 B SP1 software (Carl Zeiss). ROIs were used to measure the raw fluorescence that was then converted into relative intensity (each raw fluorescence divided by 0-h fluorescence).

Fluorescent labelling was observed with a confocal microscope (LSM710, Zeiss, Germany) equipped with a × 40 apochromatic oil immersion objective (NA: 1.2). The different analyses were conducted using the Zen 2010 BSP1 software (Zeiss, Germany). Acquisitions were performed under exactly the same conditions. The excitation wavelength for Dapi/Hoechst 33342 was set at 405 nm and collected between 421 and 517 nm. The excitation wavelength for Alexa Fluor 488 was set at 488 nm and collected between 494 and 552 nm. The excitation wavelength for Alexa Fluor 555 Streptavidin was set at 561 nm and collected between 565 and 680 nm. The pinhole aperture was set at one Airy Unit (AU). Images were processed with the Image J® software version 2.1.0 (National Institutes of Health, USA).

We stained fixed cells with 2.5 µM C11‐BODIPY581/591 (SIGMA ALDRICH, D-3861) for 60 min. The ROS-dependent oxidation of the polyunsaturated butadienyl portion of this lipid probe results in a shift of the fluorescence emission peak from ~590 nm (reduced: red) to ~510 nm (oxidized: green)^{107 ,108} . Briefly, cells were treated with 0.2 µM FASNi or 0.2% DMSO, for 4 days. In rescue experiments, cells were co-treated with FASNi and one of the following: palmitate (100 µM), phosphatidylcholine (PC, 100 µM), or Ferrostatin-1 (Fer-1, 1 µM) for 4 days. 5 mM N-acyl-cysteine (NAC) was added for 60 min after 4 days of FASNi treatment. For snap-frozen samples, we used 10 µm-thick cryo-sections of A549 xenografts (5 mice/group) and lungs of CCSP-rtTA/Tet-op-Kras (3 mice/group). Samples were incubated with C11‐BODIPY581/591, washed with PBS, fixed with 10% formalin for 1 h, counterstained with DAPI and mounted using Fluoromount-G medium (Thermo Scientific). For cells, three images per slide were acquired using a Zeiss LSM 710 confocal microscope equipped with a Plan-Apo 63x/1.4 oil DIC M27 objective and ZEN 2010 B SP1 software (Zeiss). For tissues, three images per sample were acquired using a Lionheart FX (BioTek) and Gen5 software (v3.06). We quantified the images with ImageJ software (version 1.46; NIH, Bethesda, MD, USA).

HeLa cells (American Type Culture Collection) were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% GlutaMAX and pen-strep in 24-wells plates on coverslips. Fluorescein isothiocyanate (FITC)-labelled Stx1B (10 μg/mL in 300 μL of fresh medium) was added to the cells and incubated for 1 h on 37°C. The cells were then washed with PBS, fixed with 4% paraformaldehyde (PFA) in PBS for 15 min and permeabilized with 0.1% triton X-100 in PBS for 10 min. Non-specific staining was blocked with 3% BSA in PBS for 1 h. Golgi apparatus (GA) was labelled with mouse monoclonal anti-human Golgin-97 primary antibody (0.4 μg/ml in 3% BSA for 1 h, Life Technologies, CA, USA) and with Alexa Fluor 555-conjugated donkey anti-mouse secondary antibody (1:1000 in 3% BSA for 1 h, A-31570, Life technologies). Coverslips were mounted with ProlongGold Antifade reagent with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen). Immunostained cells were visualized with LSM-710 confocal microscope (Carl Zeiss, Germany), and images were acquired and processed using ZEN 2010 B SP1 software (Carl Zeiss).

For confocal microscopy, 2.5 × 10⁵ transfected cells expressing GFP-tagged EAAT3, CFP-tagged ARFGAP1 or CFP-tagged ARF6 were seeded onto 22 mm glass bottom dishes (Invitro Scientific, Sunnyvale, CA, United States). The endoplasmic reticulum was visualized with the ER-Tracker™ Red dye (100 nM: Molecular Probes, Leiden, Netherlands) according to the manufacturer’s protocol. The plasma membrane was stained with trypan blue as described earlier (Korkhov et al., 2006 (link)). Images were captured Zeiss LSM780 equipped with an argon laser (at 30 milliwatts) and a 63x oil immersion objective (1.4 NA, Zeiss Plan-Neofluar). The images were processed using the Zen 2010 B SP1 software (Zeiss, Oberkochen, Germany).